Antithrombin is an important blood coagulation inhibition system factor (a serine protease inhibitor) in blood, which inhibits activity of a serine protease that relates to blood coagulation and the like by irreversibly forming a stable complex with a serine protease in a one molecule-to-one molecule manner. Antithrombin is mainly synthesized in liver and is present in human blood plasma in an amount of about 150 mg/l (Non-patent Reference 1).
Antithrombin is a glycoprotein comprising 432 amino acids and having a molecular weight of approximately 59,000 to 65,000, and has three disulfide bonds, Cys8-Cys128, Cys21-Cys95 and Cys247-Cys430, in its molecule. N-Glycoside-linked sugar chains are added to 4 positions, the 96th, 135th, 155th and 192nd asparagine residues counting from the N-terminus (hereinafter referred to as Asn96, Asn135, Asn155 and Asn192, respectively) of antithrombin. The antithrombin in human plasma exists in two kinds of isoforms, α-form (α type) having four N-glycoside-linked sugar chains and β-form (β type) having only three N-glycoside-linked sugar chains but not having a sugar chain to the Asn135 (Non-patent Reference 2, Non-patent Reference 3). The sugar chain N-glycoside-linked to antithrombin is a complex sugar chain comprising N-acetylglucosamine, sialic acid, galactose and mannose and has sialic acid on its terminal. The larger the binding number of sialic acids bound to the sugar chain terminal is, the longer the half-life of a glycoprotein in blood is. It is said that this is because when the sialic acid is eliminated, the galactose residue exposed to the non-reducing-end group is captured by an asialoglycoprotein (a galactose receptor localized in the liver) and quickly degraded (Non-patent Reference 4, Non-patent Reference 5). In addition, the antithrombin easily is changed into an inactive form (latent form) by a physical stress, such as heat, or a chemical stress, such as an acid (Non-patent Reference 6).
From 90 to 95% of the antithrombin in human blood plasma is composed of the α-form, and the remaining 5 to 10% is composed of the β-form (Non-patent Reference 2). In addition, about 70% of the sugar chains bound to the antithrombin in human blood plasma have two sialic acids per sugar chain, and the remaining, about 30% of the sugar chains, have one sialic acid per sugar chain (Non-patent Reference 1, Non-patent Reference 7).
The antithrombin purified from human blood plasma is used as a therapeutic agent for thrombosis based on congenital antithrombin deficiency and disseminated intravascular coagulation (DIC) which accompanies lowering of antithrombin. However, the antithrombin purified from human blood plasma has a danger of causing contamination with blood-derived viruses such as AIDS virus and human parvovirus, mutation type Creutzfeldt-Jakob disease factor, transmissible spongiform encephalopathy (TSE) factor and the like. Based on such a background, in recent years, a recombinant antithrombin has been attempted to produce from an animal cell, such as Chinese hamster ovary cell (CHO cell), or a transgenic animal, using recombinant techniques.
Unlike the human blood plasma-derived antithrombin purification product, the antithrombin obtained from an antithrombin-producing recombinant CHO cell or transgenic animal is a mixture of α-form, β-form, latent form, inactive precursor (prelatent form) or associated form of the antithrombin, host-derived impurities and the like. In addition, the number of sialic acids bound to the sugar chain terminal is also irregular (Non-patent Reference 8, Non-patent Reference 9).
As the purification method of antithrombin, heparin chromatography (Non-patent Reference 3), cation exchange chromatography (Patent Reference 1), hydroxyapatite chromatography (Patent Reference 2), hydrophilic chromatography (Non-patent Reference 8, Non-patent Reference 9, Patent Reference 3, Patent Reference 4) and the like are known. However, nothing is known about the process for preparing an antithrombin composition having a desired α-form or β-form content rate from a mixture of α-form, β-form, latent form, prelatent form and associated form.    Non-Patent Reference 1: The Second Series of Pharmaceutical Research, 20, 185 (1992)    Non-Patent Reference 2: Japanese Journal of Thrombosis and Hemostasis, 10, 93 (1999)    Non-Patent Reference 3: J. Biol. Chem., 260, 610 (1985)    Non-Patent Reference 4: Sequences of protein of immunological interest Fourth Edition, Public Health Service National Institutes of Health (1987)    Non-Patent Reference 5: Blood, 73, 84 (1987)    Non-Patent Reference 6: Structure, 2, 257 (1994)    Non-Patent Reference 7: Arch. Biochem. Biophys., 203, 458 (1980)    Non-Patent Reference 8: Protein Expression and Purification, 41, 323 (2005)    Non-Patent Reference 9: Blood, 91, 4561 (1998)    Patent Reference 1: JP-A-2003-520805    Patent Reference 2: JP-A-2003-520806    Patent Reference 3: JP Patent 2852307    Patent Reference 4: JP Patent 2678249